Dietary glucosamine overcomes the defects in αβ-T cell ontogeny caused by the loss of de novo hexosamine biosynthesis

T cell development requires the coordinated rearrangement of T cell receptor (TCR) gene segments and the expression of either αβ or γδ TCR. However, whether and how de novo synthesis of nutrients contributes to thymocyte commitment to either lineage remains unclear. Here, we find that T cell-specific deficiency in glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1), the rate-limiting enzyme of the de novo hexosamine biosynthesis pathway (dn-HBP), attenuates hexosamine levels, blunts N-glycosylation of TCRβ chains, reduces surface expression of key developmental receptors, thus impairing αβ-T cell ontogeny. GFAT1 deficiency triggers defects in N-glycans, increases the unfolded protein response, and elevates γδ-T cell numbers despite reducing γδ-TCR diversity. Enhancing TCR expression or PI3K/Akt signaling does not reverse developmental defects. Instead, dietary supplementation with the salvage metabolite, glucosamine, and an α-ketoglutarate analogue partially restores αβ-T cell development in GFAT1T-/- mice, while fully rescuing it in ex vivo fetal thymic organ cultures. Thus, dn-HBP fulfils, while salvage nutrients partially satisfy, the elevated demand for hexosamines during early T cell development.


March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. No statistical method was used to predetermine sample size.. Based on several years of working with most of our current animal models, we have taken into consideration that smaller number of cells will be obtained in GFAT1, GFAT1/PTEN and rictor knockout mice with or without transgenic TCR. Therefore, we needed more of these mice since we have done biochemical analyses as well as metabolomics, proteomics and therefore needed to obtain sufficient amount of cells. Typically the amount of thymocytes harvested from a 5 wk-old WT thymus is equivalent to~3-4 thymus from rictor-deficiency and 4-5 thymus from GFAT1-or any combination with GFAT1-deficiency (cf. Chou et all, J Immunol 2014; 193:1162-1170 and current manuscript). Experiments are typically done using 3-6 of each cohort to get statistical significance. A p value of less than 0.05 was considered significant.

No data were excluded
Each experiments were performed 2-4 times for reproducibility. All attempts at replication were successful. Glycomics experiment was only performed once on pooled samples comparing WT vs GFAT1-deficient samples.
Samples were allocated based on mouse genotyping done by PCR Blinding was not relevant for the majority of our studies since quantitative analysis was performed to compare samples. For FTOCs supplementation, analysis was blinded since genotype sof samples were only revealed after results of supplementation effects were obtained.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.